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Establishment of an in vitro platform for the characterization of proteins involved in the intestinal epithelial barrier dysfunction of Inflammatory Bowel Disease, with a focus on TNFα/TNR1A/TNR1B.

Vergara Dal Pont, Ignacio Andrés

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The gastrointestinal (GI) epithelium consists of a cellular monolayer that prevents the infiltration of pathogens and antigens into the GI mucosa and concomitantly mediates the highly selective resorption and secretion of nutrients, solutes and water. An intact monolayer of epithelial cells is achieved by two types of intercellular junctional protein complexes that seal the paracellular space: the Adherens (AJs) and the Tight Junctions (TJs). Inflammatory Bowel Disease (IBD) is a complex pathological condition originating from a disturbed homeostasis of the immune and epithelial barrier functions of the GI mucosa that results in a perturbance of the tight balance in between pro- and anti-inflammatory cytokines, and consequently in between the immune tolerance and the defensive inflammatory responses towards the microbiota and microbial antigens. The dysfunction of the GI epithelial barrier plays a major role in both the pathogenesis and the chronic manifestation of IBD. Tumor Necrosis Factor alpha (TNFα) as a key pro-inflammatory cytokine with pleiotropic functions is highly upregulated at the intestinal mucosa in IBD. The TNFα signalling cascade is initiated by two different ligands, the membrane (mTNFα) and the soluble (sTNFα) forms of TNFα, and mediated by two different membrane receptors TNR1A (TNFRSF1A) and TNR1B (TNFRSF1B). The latter are not only activated in separate ways, but can determine divergent effects in a tissue- and cell-specific manner. In my Ph.D. work, I aimed to: i) establish an in vitro platform for the characterization of candidate genes/proteins with regards to the key mechanisms that regulate the paracellular permeability at the GI epithelium, ii) establish an inflammatory model to resemble the barrier dysfunction that takes place in IBD, iii) investigate the mechanisms of action of the key pro-inflammatory cytokines TNFα, Interferon gamma (IFNγ) and Interleukin1- beta (IL-1β), with a special focus on the dissection of the axis TNFα/TNR1A/TNR1B, in intestinal epithelial barrier function. An in vitro model of the inflamed intestinal epithelium was generated by differentiating the colorectal carcinoma cell lines T84 and CACO-2 on Transwell inserts and applying the pro-inflammatory cytokines sTNFα, IFNγ and IL-1β from the basolateral side at different concentrations and combinations. To assess the consequences of these stimuli on the different permeability pathways of the paracellular route, different assays were performed: measurement of the Trans Epithelial Electrical Resistance (TEER), permeability assays with fluorescently labelled molecular species, cell viability assays and assessment of the expression and subcellular localization of key tight junctional proteins (TJPs). Once defined the optimal pro-inflammatory input to mimic the IBD condition, the barrier function impairing effects of the single cytokines sTNFα and IFNγ were investigated in detail. Fully differentiated T84 monolayers were subjected to a single cytokine induction in the presence of potent and selective blockers of both the ligands and of the respective cognate membrane receptors to confirm the validity of the model. A part from applying the key functional and cell viability assays, the cross-regulatory mechanisms taking place in between both cytokines were investigated with transcriptional expression analyses. The results of these experiments confirmed a synergism in between both cytokines based on not only a positive regulatory loop exerted by IFNγ on TNFRSF1A and TNFRSF1B, but also on a positive regulatory loop exerted by sTNFα on IFNGR1 and IFNGR2. Furthermore, a putative anti-necroptotic effect determined by IFNγ was observed to take place on these GI epithelial cells. In the last part of my work, I applied potent and selective pharmacological modulators directed towards different levels of the TNFα signalling. The colorectal carcinoma cell line T84 was fully differentiated on Transwell inserts and different end-point experiments were run by applying combinations of sTNFα (natural agonist of TNR1A and marginal agonist of TNR1B), TROS (competitive, selective antagonist of TNR1A) and ADALIMUMAB (competitive, global antagonist of the sTNFα signalling). The consequences of these modulations were analyzed with the different aforementioned functional assays, and a detailed image of the cellular pathways differentially regulated was obtained by a transcriptomic expression analysis. The results of these experiments demonstrated that TNR1A mediates most of the barrier function impairment determined by sTNFα. The complete dissection of the signalling, by future investigation of the role of TNR1B in this specific context, could provide the basis for the initiation of the work towards the modification of the actual pharmacological therapies for IBD that are dominated by global TNFα inhibitors. A receptor-specific therapy could lead to a better outcome rather than the neutralization of the whole signalling pathway.

Document type: Dissertation
Supervisor: Rappold, Prof. Dr. Gudrun
Place of Publication: Heidelberg
Date of thesis defense: 6 October 2022
Date Deposited: 23 Nov 2022 09:49
Date: 2022
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
DDC-classification: 570 Life sciences
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